DESCRIPTION: The activation of dioxygen in biological systems is a question of great importance because of its involvement in many metabolic processes. Although the mechanism of oxygen activation in heme systems such as cytochrome P450 and peroxidases is becoming quite well understood, much less is known of he corresponding mechanisms for non-heme systems. These latter enzymes are involved in the biosynthesis of amino acids, neurochemicals, antibiotics, and prostaglandin precursors and participate in reactions that range from oxidative cleavage of aromatic rings, aliphatic and aromatic hydroxylation, to ring cyclizations involving heteroatoms. For many mononuclear nonheme iron enzymes, the reaction with O2 occurs only after substrate or cofactor interacts with the active site iron, e.g. catechol for catechol dioxygenases, substrate thiol for isopenicillin N synthase, and the a-keto acid cofactor for prolyl hydroxylase, clavarninate synthase and other a-ketoglutarate-dependent oxygenases. It is proposed that a common general mechanism unites the chemistry of these enzymes, but the details of the dioxygen activation step vary from enzyme to enzyme because of the divergent natures of the substrates and/or cofactors. Important questions include, 1) how is O2 activated by these enzymes, and 2) what is the nature of high valent intermediates that are involved in the reactions? Spectroscopic (NMR, EPR, resonance Raman, Mossbauer, EXAFS, electrospray mass spectrometry) and mechanistic studies are proposed for a number of enzymes to elucidate details of the active site structure and the mechanism of action. These include intradiol and extradiol cleaving catechol dioxygenases and the a-keto acid dependent enzymes, 2,4- diclorophenoxyacetate/a- ketoglutarate dioxygenase and clavaminate synthase. Model complexes will be synthesized and used to interpret spectroscopic features of the enzymes and mimic the key mechanistic steps of the oxidations. Modeling efforts will focus on: 1) extradiol cleavage of catechols, 2) the role the a- keto acid cofactor plays in oxygenase reactions, 3) the role of the Fe(III)-OH moiety in the lipoxygenase reaction, and 4) characterization of transient iron-peroxo and iron-oxo intermediates. As in the past, the synergistic interaction of the biochemical and inorganic aspects of the proposal is important for the success of the program.